The disclosed subject matter provides techniques for the generation of microtissues within three-dimensional constructs for uses including screening applications.
Heart failure imposes a major public health burden that is inadequately addressed by current approaches, striking one of every five Americans and accounts annually for approximately one million hospitalizations, over 50,000 deaths, and almost $35 billion in health care costs.
One approach to understanding the pathogenesis of heart failure can include using manipulating individual genes in animal models. Certain research in this area has implicated a variety of individual molecules, signaling pathways and gene programs in the myocardial response to injury and overload. Despite these preclinical advances, few of these regulators have been rigorously assessed in the human heart, and translation into an effective heart failure therapy remains merely an aspiration—pharmacologic therapy for heart failure has changed little over the past 15 years. Contributors to this “translational divide” in agents targeting the heart may include, for example, shortcomings of existing animal models and the absence of suitable in vitro models that reflect the biology of the human myocardium.
Although human induced pluripotent stem cell (iPSC) derived myocytes have demonstrated patient-specific electrophysiological abnormalities (e.g., action potential prolongation in myocytes from patients with the long-QT syndrome due to KCNQ1 mutation), iPSC-derived myocytes can fail to recapitulate certain of the critical functions of tissue when they are cultured using traditional 2D culture substrates. The adhesion of cells to a rigid, planar substrate can be structurally and mechanically non-physiologic. For example, cells can assume a flattened morphology with an artificial introduction of apical-basal polarity of cell-matrix adhesions, and cell-cell adhesions can be unable to bear substantial mechanical load owing to the rigid constraint of the underlying substrate. As a result, the architecture of these cells can differ from those of in vivo tissue. Thus, certain aspects of physiology and pathology involving contractile and structural responses can be unmet by traditional 2D approaches.
Characteristic physiological and pathological responses to biomechanical load, transduced through complex three-dimensional tissue architecture, are fundamental features of the myocardium. Variations in preload and afterload can alter myocardial contractility on a beat-to-beat basis and induce changes in myocardial gene expression and growth over time. Conversely, the absence of relevant biomechanical cues or a multicellular, three-dimensional (3D) architecture can distort the phenotype of in vitro myocardial models. Thus, myocytes adapted to conventional two-dimensional cell culture models can differ from those of freshly isolated myocytes from the intact heart. Developmental and species differences can further exacerbate differences between adult human myocardium and conventionally used neonatal rodent myocyte cultures. Together, these realities can compromise the clinical relevance of efforts to use cultured myocyte models in basic, translational and preclinical pharmacological research.
Additionally, these issues are not limited to cardiac testing, as many other tissues and biological processes are faced with similar concern, such as high cost, ethical concerns, and experimental limitations of animal models. For example, certain techniques used in connection with other tissues can likewise fail to recapitulate biological functions of the tissues. The use of stem cell technologies can produce useful sources of human cells. These cells can, in some cases, regain certain in vivo functions in an appropriate in vitro environment. However, certain techniques for generating tissues from these cells in an in vitro environment can fail to provide clinically relevant culture models.
Accordingly, there is a need for improved techniques for the generation of microtissues, including cardiac microtissues.